Abstract

The free hormone hypothesis postulates that only the nonbound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert biologic effects. In this review, I will examine four hormone groups-vitamin D metabolites (especially 25OHD), thyroid hormones (especially thyroxine [T4]), sex steroids (especially testosterone), and glucocorticoids (especially cortisol)-that are bound to various degrees to their respective binding proteins-vitamin D-binding protein (DBP), thyroid-binding globulin (TBG), sex hormone-binding globulin (SHBG), and cortisol-binding globulin (CBG)-for which a strong case can be made that measurement of the free hormone level provides a better assessment of hormonal status than the measurement of total hormonal levels under conditions in which the binding proteins are affected in levels or affinities for the hormones to which they bind. I will discuss the rationale for this argument based on the free hormone hypothesis, discuss potential exceptions to the free hormone hypothesis, and review functions of the binding proteins that may be independent of their transport role. I will then review the complications involved with measuring the free hormone levels and the efforts to calculate those levels based on estimates of binding constants and levels of both total hormone and total binding protein. In this review, the major focus will be on DBP and free 25OHD, but the parallels and differences with the other binding proteins and hormones will be highlighted. Vitamin D and its metabolites, thyroid hormones, sex steroids, and glucocorticoids are transported in blood bound to serum proteins. The tightness of binding varies depending on the hormone and the binding protein such that the percent free varies from 0.03% for T4 and 25OHD to 4% for cortisol with testosterone at 2%. Although the major function of the primary carrier proteins (DBP, TBG, SHBG, and CBG) may be to transport their respective lipophilic hormones within the aqueous media that is plasma, these proteins may have other functions independent of their transport function. For most tissues, these hormones enter the cell as the free hormone presumably by diffusion (the free hormone hypothesis), although a few tissues such as the kidney and reproductive tissues express megalin/cubilin enabling by endocytosis protein-bound hormone to enter the cell. Measuring the free levels of these protein-bound hormones is likely to provide a better measure of the true hormone status than measuring the total levels in situations where the levels and/or affinities of the binding proteins are altered. Methods to measure free hormone levels are problematic as the free levels can be quite low, the methods require separation of bound and free that could disturb the steady state, and the means of separating bound and free are prone to error. Calculation of free levels using existing data for association constants between the hormone and its binding protein are likewise prone to error because of assumptions of linear binding models and invariant association constants, both of which are invalid. © 2020 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.


Authors

Bikle, Daniel D

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  • 1 reviewer
  • pre-publication peer review (FINAL ROUND)
    Decision Letter
    2020/09/22

    22-Sep-2020

    Dear Dr. Bikle:

    It is a pleasure to accept your manuscript entitled "The free hormone hypothesis: when, why, and how to measure the free hormone levels to assess vitamin D, thyroid, sex hormone, and cortisol status." in its current form for publication in JBMR Plus. If there were further comments from the reviewer(s) who read your manuscript, they will be included at the foot of this letter.

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    Deputy Editor Comments to Author:

    Adding a comment as recommended by a reviewer would not change the overall message of the manuscript, which is dealing with "free hormone secretion". Thus, the presence of DBP in the alpha cells does not change the overall message as currently presented by the authors.

    Reviewer(s)' Comments to Author:

    Reviewer: 2

    Comments to the Author
    This is a nice review article but I think it just needs a couple of sentences to mention the important role that DBP plays in actin-binding. For years this was thought to occur in the general circulation, but a recent Cell Reports paper shows that this can occur intracellular
    https://www.cell.com/cell-reports/pdfExtended/S2211-1247(20)30741-5
    Recognition of this would truly bring the review up to date

    Decision letter by
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    Reviewer report
    2020/09/16

    This is a nice review article but I think it just needs a couple of sentences to mention the important role that DBP plays in actin-binding. For years this was thought to occur in the general circulation, but a recent Cell Reports paper shows that this can occur intracellular
    https://www.cell.com/cell-reports/pdfExtended/S2211-1247(20)30741-5
    Recognition of this would truly bring the review up to date

    Reviewed by
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  • pre-publication peer review (ROUND 1)
    Decision Letter
    2020/08/05

    05-Aug-2020

    RE: JBMR Plus MS# JBM4-07-20-0085 : The free hormone hypothesis: when, why, and how to measure the free hormone levels to assess vitamin D, thyroid, sex hormone, and cortisol status.

    Dear Dan:

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    Deputy Editor Comments to Author:

    Good, concise review, especially by comparison with other transport protein reviews.

    Reviewer(s)' Comments to Author:

    Reviewer: 1

    Comments to the Author
    1. The next to the last line in paragraph 1 of the “The free hormone hypothesis” section should read “total hormone”, not just hormone.
    2. In the second paragraph of this section the authors should supplement the Mendel hypothesis by stating that it is the rate of blood and/or urine flow that may affect the free hormone hypothesis.
    3. In line 11 of the section “The megalin/cubulin transport” the authors should address the situation with megalin/cubulin in the context of the disease-activated macrophage along with the other two extra-renal 1,25-dihydroxyvitamin D synthetic sites mentioned, the parathyroid gland and placenta.
    4. In the first paragraph of the “DPB and free vitamin D metabolites” section the authors should mention that DBP is a member of the albumin super family of proteins and perhaps provide a cartoon of the functional structural domains of DBP and albumin; this will be important when discussing the membrane binding domain of DBP as well as the other binding proteins and albumin.
    5. In the 3rd paragraph of the “Structure and polymorphisms” section the authors should expand on the potential purpose of the membrane binding domains in DBP. For example, are they there to anchor DBP to the cell surface in order to reduce the diffusion distance for 25-hydroxyvitamin D (25D) to get into the cells? Is this the reason why the percentage of free 25D in the presence of DBP is so low compared to albumin and the other known binding proteins? Are there mutations found in either of these membrane interaction domains that would shed light on their functional significance?
    6. In line 16 of the “Summary/Conclusion” section, the authors might add the macrophage activating factor (MAF) potential of DBP as a non-ligand-determined role along with actin binding.

    Decision letter by
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    Reviewer report
    2020/07/22

    1. The next to the last line in paragraph 1 of the “The free hormone hypothesis” section should read “total hormone”, not just hormone.
    2. In the second paragraph of this section the authors should supplement the Mendel hypothesis by stating that it is the rate of blood and/or urine flow that may affect the free hormone hypothesis.
    3. In line 11 of the section “The megalin/cubulin transport” the authors should address the situation with megalin/cubulin in the context of the disease-activated macrophage along with the other two extra-renal 1,25-dihydroxyvitamin D synthetic sites mentioned, the parathyroid gland and placenta.
    4. In the first paragraph of the “DPB and free vitamin D metabolites” section the authors should mention that DBP is a member of the albumin super family of proteins and perhaps provide a cartoon of the functional structural domains of DBP and albumin; this will be important when discussing the membrane binding domain of DBP as well as the other binding proteins and albumin.
    5. In the 3rd paragraph of the “Structure and polymorphisms” section the authors should expand on the potential purpose of the membrane binding domains in DBP. For example, are they there to anchor DBP to the cell surface in order to reduce the diffusion distance for 25-hydroxyvitamin D (25D) to get into the cells? Is this the reason why the percentage of free 25D in the presence of DBP is so low compared to albumin and the other known binding proteins? Are there mutations found in either of these membrane interaction domains that would shed light on their functional significance?
    6. In line 16 of the “Summary/Conclusion” section, the authors might add the macrophage activating factor (MAF) potential of DBP as a non-ligand-determined role along with actin binding.

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